1. Field of the Invention
Embodiments of the invention are directed generally to methods for preparing, purifying, amplifying, and detecting biological molecules of interest such as nucleic acids and, more particularly, to such methods performed using microfluidic devices.
2. Description of the Related Art
The use of molecular diagnostics has expanded greatly since its inception in the early 1980s, particularly as a means to permit the detection of slow growing or fastidious bacteria responsible for infectious diseases. The detection of viral pathogens, including viral load testing has also been significantly improved by molecular diagnostics. As more data have become available regarding the human genome, the use of molecular diagnostics in pharmacogenomics, companion diagnostics, and other personalized medicine applications continues to gain momentum.
Nucleic acid amplification is a standard technique known in the art by which nucleic acids may be isolated and more accurately and efficiently manipulated for use in molecular diagnostics and other nucleic acid screening purposes. Various methods have been described in the literature for amplifying nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), present in a sample. Among these, the most widely practiced is the polymerase chain reaction (PCR), described in U.S. Pat. No. 4,683,195 (Mullis et al., entitled “Process for amplifying, detecting, and/or -cloning nucleic acid sequences,” issued Jul. 28, 1987) and U.S. Pat. No. 4,683,202 (Mullis, entitled “Process for amplifying nucleic acid sequences,” issued Jul. 28, 1987). Briefly, PCR consists of amplifying denatured, complementary strands of target nucleic acid by annealing each strand to a short oligonucleotide primer, wherein the primers are chosen so as to flank the sequence of interest. The primers are then mediated by a polymerase enzyme to yield extension products that are themselves complementary to the primers and hence serve as templates for synthesis of additional copies of the target sequence. Each successive cycle of denaturation, primer annealing, and primer extension essentially doubles the amount of target synthesized in the previous cycle, resulting in exponential accumulation of the target.
PCR methodologies in general suffer from several limitations that are well-known in the art. One such limitation is owing to the poor fidelity of commonly used, thermostable polymerase enzymes, such as Taq. This results in nucleotide base misincorporations that are propagated from one cycle to the next. It is estimated that such misincorporations may occur as often as once per one thousand bases of incorporation. A second limitation is that different cDNAs are amplified with different efficiencies, resulting in underrepresentation of some cDNA sequences and overrepresentation of others in the amplified product. Even a small difference in efficiency may result in a several-thousand fold differential in the representation of these cDNAs in the product after only as few as 30 cycles of amplification.
Another limitation is the presence of inhibitors of PCR in the starting material (e.g., hemoglobin in blood). These inhibitors are often carried through purification and either limit or completely impede amplification reactions performed with the nucleic acids derived from the purification. Therefore this provides another rationale for needing better upstream purification strategies (see e.g., J. Bessetti, Profiles in DNA, PCR Inhibition, An Introduction to PCR Inhibitors, Promega Corporation, March 2007).
Also significant is that nucleic acid amplification is only as accurate as the starting sample of nucleic acid to be amplified. Nucleic acid (e.g., DNA or RNA) of low purity will yield amplification products that may not reflect the composition of the starting sample, and therefore, cannot be used (or relied on) for diagnostic purposes.
It is also recognized that the conventional practice of biochemistry and molecular biology can require physical process resources on a scale that are frequently inversely proportional to the size of the subject being studied. For example, the apparatus and process chemistry associated with the preparation and purification of a biological sample such as a nucleic acid fragment for prospective analysis may easily require a full scale bio-laboratory with sterile facilities. Furthermore, an environmentally isolated facility of similar scale may typically be required to carry out the known nucleic acid amplification procedures such as polymerase chain reaction (PCR) for amplifying the nucleic acid fragment.
There is therefore a need in the art for improved methods of nucleic acid purification for use in nucleic acid amplification techniques, so that nucleic acids may be more accurately and efficiently purified, identified and manipulated for use in molecular diagnostics and other nucleic acid screening purposes. There also exists a need for improved microfluidic systems for processing fluids for analysis of biological or chemical samples, and in particular, in the detection and analysis of biologically active macromolecules derived from such samples such as DNA, RNA, amino acids and proteins. It is beneficial and advantageous that the systems are mass producible, inexpensive, and preferably disposable; that the systems be simple to operate and that many or substantially all of the fluid processing steps be automated; that the systems be customizable, and be modular such that the system can be easily and rapidly reconfigured to suit various applications in which the detection of macromolecules is desired; and, that the systems be able to provide straightforward and meaningful assay results. A more thorough discussion of the challenges and shortcomings in the art, as well as exemplary solutions that may be utilized in conjunction with the teachings of the instant invention, are disclosed in applicant's copending U.S. application Ser. No. 13/033,165 (Pub. No. US2011/0275058) entitled “Self-contained Biological Assay Apparatus, Methods, and Applications,” the subject matter of which is incorporated herein by reference in its entirety.